The present invention relates to a 5-substituted hydantoin racemase, which efficiently catalyzes racemization reactions at a high optimum temperature for racemization reactions, DNA coding for the racemase, and processes for producing optically active amino acids.
The 5-substituted hydantoin racemase enzyme (hereinafter abbreviated to HRase) catalyzes a racemization reaction of an optically active 5-substituted hydantoin compound, i.e., a D- or L-5-substituted hydantoin compound.
The 5-substituted hydantoin compound undergoes a hydrolysis reaction with hydantoinase (1) and Carbamyl amino acid hydrolase (2) to form an amino acid as shown in the following reaction scheme (I): 
As shown in the reaction scheme above, the hydantoinase hydrolyzes the 5-substituted hydantoin compound thereby forming N-carbamyl amino acid. Additionally, the N-carbamyl amino acid hydrolase hydrolyzes N-carbamyl amino acid thereby forming an optically active amino acid. These enzymes should be optically selective.
Methods of using a microbial enzyme system and combining a bacterial enzyme system with a chemical reaction system have been previously described. Production of optically amino acids from 5-substituted hydantoin compounds is important in the production of pharmaceutical preparations, chemical industry products, food additives and other similar articles or products.
The optico-selective hydrolysis should enable the efficient racemization of one enantiomer to another enantiomer, the second enantiomer can further serve as a substrate, for example see the above reaction scheme. Furthermore, this conversion can be performed using microbial enzymes or microbial enzymes combined with a chemical reaction system. Under neutral conditions where the enzyme system is active, the racemization of the optically active 5-substituted hydantoin compound, not serving as the substrate, is very low. Therefore, the racemization becomes rate-determining and resulting in poor conversion into the optically active amino acid.
Accordingly, for the purpose of racemization of the optically active 5-substituted hydantoin compound under neutral conditions, HRase was searched for, and HRases derived from microorganisms of the genus Arthrobacter (Japanese Patent Application Laid-Open (JP-A) No. 62-122591; Ann. N.Y. Acad. Sci., 672, 478; Japanese Patent Application Laid-Open (JP-A) No. 6-343462) and microorganisms of the genus Pseudomonas (Japanese Patent Application Laid-Open (JP-A) No. 4-271784; J. Bacteriol., 174, 7989 (1992)) have been reported. The optimum reaction temperatures of the previously reported HRases derived from microorganisms are 37xc2x0 C. for the enzyme derived from Arthrobacter sp. DSM-3747 (Ann. N.Y. Acad. Sci., 672, 478), 10 to 50xc2x0 C. for the enzyme derived from Arthrobacter sp. DK200 (Japanese Patent Application Laid-Open (JP-A) No. 62-122591), and 45xc2x0 C. for the enzyme derived from Pseudomonas sp. NS671.
Generally, when the working optimum temperature of an enzyme is increased, the industrial utility value of the enzyme is also increased. That is, if the reaction temperature can be increased, the reaction rate can be also increased, which results not only in efficient progress of the desired reaction but also in a reduction in the risk of contamination of the reaction solution with microorganisms during the reaction. This results in several advantages including easy process and quality control.
As described above, HRase having a higher optimum temperature for the reaction is useful from the viewpoint of industrial applicability. However, the upper limit of the working optimum temperatures of the previously reported HRases is 50xc2x0 C. and an HRase having a higher working optimum temperature is desired to achieve efficient progress of the racemization reaction and to reduce the risk of contamination of the reaction solution with microorganisms.
It is an object of the present invention to isolate a novel HRase having a higher reaction optimum temperature than conventional and to provide a process for producing an optically active amino acid by use of the enzyme.
As a result of extensive study in view of the problem described above, the present inventors found a novel HRase having a desired higher reaction optimum temperature is present in a Microbacterium microorganism, thus arriving at completion of the present invention.
One object of the present invention, is providing a new process adjuvant for improving the racemization reaction of 5-substituted hydantoin compounds and optically active amino acids.
Another object of the invention is to provide a nucleotide sequence encoding a polypeptide which has HRase activity. One embodiment of such a sequence is the nucleotide sequence of SEQ ID NO: 1. Other embodiments include nucleotide sequences that are complimentary to the sequences described herein, those sequences that are 70%, 80% and/or 90% identical to the sequences described herein and/or those sequences that hybridize under stringent conditions to the sequences described herein.
A further object of the invention is a method of making HRase or an isolated polypeptide having a HRase activity, as well as use of such isolated polypeptides in the production of amino acids. One embodiment of such a polypeptide is the polypeptide having the amino acid sequence of SEQ ID NO: 2. Other embodiments are this polypeptide are those that are 70%, 80% and/or 90% identical to the amino acid sequences described herein. Another embodiment of the polypeptide is that which has racemase activity with an optimal working pH of from about 7 to about 9 and an optimal working temperature of from about 50 to about 60xc2x0 C.
Other objects of the invention include methods of detecting nucleic acid sequences homologous to the sequences described herein, particularly nucleic acid sequences encoding polypeptides that have HRase activity, and methods of making nucleic acids encoding such polypeptides.
Another object of the present invention is to provide methods of preparing optically active hydantoin compounds by contacting a 5-substituted hydantoin compound with the HRase described herein.
Another object of the present invention is to provide methods of preparing an N-carbamyl amino acid by contacting a 5-susbstituted hydantoin compound with the HRase described herein and an enzyme that will hydrolyze the 5-substituted hydantoin compound in an optically selective manner. One embodiment of an enzyme that will hydrolyze the 5-substituted hydantoin compound is the Hydantoinase described herein. The Hydantoinase may have the amino acid sequence of SEQ ID NO:4 or amino acid sequences with substantial identity to SEQ ID NO:4 and having the hydantoinase activity.
Another object of the present invention is to provide methods of producing optically active amino acids by contacting a 5-substituted hydantoin compound with the HRase described herein, an enzyme that will hydrolyze the 5-substituted hydantoin compound in an optically selective manner, and an enzyme that will hydrolyze a N-carbamyl amino acid in an optically selective manner. One embodiment of an enzyme that will hydrolyze the 5-substituted hydantoin compound is the Hydantoinase described herein. The Hydantoinase may have the amino acid sequence of SEQ ID NO:4 or amino acid sequences with substantial identity to SEQ ID NO:4 and having the hydantoinase activity. One embodiment of the enzyme that will hydrolyze the N-carbamyl amino acid is the carbamyl amino acid hydrolase described herein. The carbamyl amnio acid hydrolase may have the amino acid sequence of SEQ ID NO:6 or amino acid sequences with substantial identity to SEQ ID NO:6 and having the hydrolase activity.
The above objects highlight certain aspects of the invention. Additional objects, aspects and embodiments of the invention are found in the following detailed description of the invention.